Excitation-contraction (EC) coupling in skeletal muscle depends upon trafficking of Ca V 1.1, the principal subunit of the dihydropyridine receptor (DHPR) (L-type Ca 2+ channel), to plasma membrane regions at which the DHPRs interact with type 1 ryanodine receptors (RyR1) in the sarcoplasmic reticulum. A distinctive feature of this trafficking is that Ca V 1.1 expresses poorly or not at all in mammalian cells that are not of muscle origin (e.g., tsA201 cells), in which all of the other nine Ca V isoforms have been successfully expressed. Here, we tested whether plasma membrane trafficking of Ca V 1.1 in tsA201 cells is promoted by the adapter protein Stac3, because recent work has shown that genetic deletion of Stac3 in skeletal muscle causes the loss of EC coupling. Using fluorescently tagged constructs, we found that Stac3 and Ca V 1.1 traffic together to the tsA201 plasma membrane, whereas Ca V 1.1 is retained intracellularly when Stac3 is absent. Moreover, L-type Ca 2+ channel function in tsA201 cells coexpressing Stac3 and Ca V 1.1 is quantitatively similar to that in myotubes, despite the absence of RyR1. Although Stac3 is not required for surface expression of Ca V 1.2, the principle subunit of the cardiac/brain L-type Ca 2+ channel, Stac3 does bind to Ca V 1.2 and, as a result, greatly slows the rate of current inactivation, with Stac2 acting similarly. Overall, these results indicate that Stac3 is an essential chaperone of Ca V 1.1 in skeletal muscle and that in the brain, Stac2 and Stac3 may significantly modulate Ca V 1.2 function.Stac adaptor protein | L-type Ca 2+ channel | excitation-contraction coupling

Skeletal muscle contraction is triggered by Ca release from the sarcoplasmic reticulum (SR) in response to plasma membrane (PM) excitation. In vertebrates, this depends on activation of the RyR1 Ca pore in the SR, under control of conformational changes of Ca1.1, located ∼12 nm away in the PM. Over the last ∼30 y, gene knockouts have revealed that Ca1.1/RyR1 coupling requires additional proteins, but leave open the possibility that currently untested proteins are also necessary. Here, we demonstrate the reconstitution of conformational coupling in tsA201 cells by expression of Ca1.1, β1a, Stac3, RyR1, and junctophilin2. As in muscle, depolarization evokes Ca transients independent of external Ca entry and having amplitude with a saturating dependence on voltage. Moreover, freeze-fracture electron microscopy indicates that the five identified proteins are sufficient to establish physical links between Ca1.1 and RyR1. Thus, these proteins constitute the key elements essential for excitation-contraction coupling in skeletal muscle.

Stromal interacting molecule (STIM) and Orai proteins constitute the core machinery of store-operated calcium entry. We used transmission and freeze-fracture electron microscopy to visualize STIM1 and Orai1 at endoplasmic reticulum (ER)-plasma membrane (PM) junctions in HEK 293 cells. Compared with control cells, thin sections of STIM1-transfected cells possessed far more ER elements, which took the form of complex stackable cisternae and labyrinthine structures adjoining the PM at junctional couplings (JCs). JC formation required STIM1 expression but not store depletion, induced here by thapsigargin (TG). Extended molecules, indicative of STIM1, decorated the cytoplasmic surface of ER, bridged a 12-nm ER-PM gap, and showed clear rearrangement into small clusters following TG treatment. Freeze-fracture replicas of the PM of Orai1-transfected cells showed extensive domains packed with characteristic "particles"; TG treatment led to aggregation of these particles into sharply delimited "puncta" positioned upon raised membrane subdomains. The size and spacing of Orai1 channels were consistent with the Orai crystal structure, and stoichiometry was unchanged by store depletion, coexpression with STIM1, or an Orai1 mutation (L273D) affecting STIM1 association. Although the arrangement of Orai1 channels in puncta was substantially unstructured, a portion of channels were spaced at ∼15 nm. Monte Carlo analysis supported a nonrandom distribution for a portion of channels spaced at ∼15 nm. These images offer dramatic, direct views of STIM1 aggregation and Orai1 clustering in store-depleted cells and provide evidence for the interaction of a single Orai1 channel with small clusters of STIM1 molecules.SOCE | STIM1 | Orai1 | nanoscale patterning | electron microscopy S pecialized junctions linking the endoplasmic reticulum (ER) to the plasma membrane (PM) were first described by Porter and Palade (1) in skeletal and cardiac muscle. In skeletal muscle, excitation-contraction coupling is mediated by direct physical contact between voltage-gated Ca 2+ channels (dihydropyridine receptors) in invaginated transverse tubules of the PM and Ca 2+

It is known that cardiac myocytes contain three categories of calcium release units (CRUs) all bearing arrays of RyR2: peripheral couplings, constituted of an association of the junctional SR (jSR) with the plasmalemma; dyads, associations between jSR and T tubules; internal extended junctional jSR (EjSR)/corbular jSR that is not associated with plasmalemma/T tubules. The bird hearts, even if fast beating (e.g., in finch and hummingbird) have no T tubules, despite fiber sizes comparable to those of mammalian ventricle, but are rich in EjSR/corbular SR. The heart of small lizard also lacks T tubule, but it has only peripheral couplings and compensates for lack of internal CRUs by the small diameter of its cells. We have extended previous information on chicken heart to finch and lizard by establishing a spatial relationship between RyR2 clusters in jSR of peripheral couplings and clusters of intra-membrane particles identifiable as voltage sensitive calcium channels (CaV1.2) in the adjacent plasmalemma. This provides the structural basis for initiation of the heart beat in all three species. Further we evaluated the distances separating peripheral couplings from each other and between EjSR/corbular SR sites within the bird muscles in all three hearts. The distances suggest that peripheral coupling sites are most likely to act independently of each other and that a calcium wave-front propagation from one internal CRU site to the other across the level of the Z line, may be marginally successful in the chicken, but certainly very effective in the finch.

Background:Calsequestrin is essential to keep a high calcium concentration inside the sarcoplasmic reticulum of muscle fibers. Results: In situ, calsequestrin polymers appear to form a three-dimensional structure with repeated nodal points. Conclusion: A three-dimensional calsequestrin polymer matrix is very suitable for its spatially confined calcium storage function. Significance: The calsequestrin structure has been extensively studied in ex vivo systems. This approach illustrates the behavior of the protein while still in its physiological cell localization.